BRAIN TUMORS CURRENT AND EMERGING THERAPEUTIC STRATEGIES doc

Judging from the research and development process, human beings have, after a hundred years’ efforts, moved from the development and application of animal models of spontaneous and induc

BRAIN TUMORS - CURRENT AND EMERGING THERAPEUTIC STRATEGIES

Edited by Ana Lucia Abujamra

Brain Tumors - Current and Emerging Therapeutic Strategies

Edited by Ana Lucia Abujamra

Published by InTech

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Contents

Preface IX

Part 1 Tumor Models, Molecular Mechanisms and Diagnostics 1

Xenograft Model of Human Brain Tumor 3

Chapter 1

Chen Hua, Dong Jun and Huang Qiang

Experimental Brain Tumors: Current Concepts 21

Chapter 2

Jesús Vaquero and Mercedes Zurita

Molecular Diagnostics of Brain Tumours

Chapter 3

by Measuring the 5-Methylcytosine Level in Their DNA 37

Anna-Maria Barciszewska, Stanisław Nowak,

Iwona Gawrońska and Mirosława Barciszewska

The Bevacizumab “Pseudoresponse”

Evolvement of Molecular Biomarkers

Chapter 8

in Targeted Therapy of Malignant Gliomas 117 Erica Hlavin Bell, Mersiha Hadziahmetovic and Arnab Chakravarti

Part 2 Chemotherapy and Other Therapeutic Strategies 143

Glioblastoma: Current Chemotherapeutic

Chapter 9

Status and Need for New Targets and Approaches 145

Aditi Jain, James CK Lai, Golam MI Chowdhury,

Kevin Behar and Alok Bhushan

Chemotherapy of Medulloblastoma in Children 177

Chapter 10

Dezső Schuler, Péter Hauser and Miklós Garami

New Therapeutic Strategies

Antiproliferative Effect of EGFR Inhibition

in Human Glioblastoma Multiforme Cell Lines 245 Georg Karpel-Massler and Marc-Eric Halatsch

Gerardo Caruso, Mariella Caffo, Giuseppe Raudino,

Concetta Alafaci and Francesco Tomasello

Cellular Immunotherapy

Chapter 18

for Malignant Brain Tumors 307 Catherine Flores and Duane A Mitchell

Immunotherapeutic Strategies for Brain Tumors 331

Chapter 19

Mitsugu Fujita and Hideho Okada

Immunotherapy with Dendritic Cells and

Chapter 20

Newcastle Disease Virus in Glioblastoma Multiforme 355

Thomas Neßelhut, Dagmar Marx,

Jan Neßelhut and Fred Fändrich

Targeted Therapy for Gliomas:

Chapter 21

the Oncolytic Virus Applications 375

Zulkifli Mustafa, Hidayah Roslan

and Jafri Malin Abdullah

cor-With this in mind, the book is divided into three sections: The first section addresses the tumor models currently available to study brain tumors, new diagnostic methods and the molecular mechanisms that are frequently involved in the pathogenesis of brain tumors Established and novel therapeutic strategies are described in section two, with a special focus of gene therapy and immunotherapy in section three

The main focus of this book is the gliomas, given their incidence and dismal prognosis, but other brain tumors, including medulloblastoma and brain metastases, are also dis-cussed Written by experts in brain tumor research and in managing brain tumors, this book provides the most up-to-date information regarding tumor models, clinical diag-nostic methods and therapeutic strategies

The chapters here presented aim in contributing to the understanding of brain tumor pathogenesis, tumor behavior within the cellular niche, and of the new therapeutic methods for managing brain tumors All topics possess translational potential in the hopes that the health care professional in the field can benefit not only by increasing knowledge, but increasing clinical applicability as well

Sincerely,

Dr Ana Lucia Abujamra

Hospital de Clínicas de Porto Alegre Oncologia Pediátrica- Terceiro Leste

R Ramiro Barcelos, 2350 Bairro Rio Branco Porto Alegre, Rio Grande do Sul 90035-903

Brasil

Tumor Models, Molecular Mechanisms and Diagnostics

Xenograft Model of Human Brain Tumor

Chen Hua1,2, Dong Jun1 and Huang Qiang1

1Department of Neurosurgery, 2nd Affiliated Hospital of Suzhou University

2Department of Neurosurgery, Nanjing First Hospital, Nanjing Medical University

China

1 Introduction

In the life sciences, scientists are paying more and more attention to the human brain, the essential center of the body The gliomas in human brains are especially unique challenges for doctors due to the difficulties of identifying early cancer lesions and little effective treatments Gliomas account for about half of the central nervous system tumors Due to its invasive growth and other malignant behaviors, gliomas are difficult to be radically cured; moreover, most of gliomas are difficult to be early detected, even they are discovered, it is difficult to cure them because of their resistance to radiation or chemotherapy Therefore, developing animal models of human gliomas is essential for us to explore the mechanisms

of occurrence and development of brain tumor and promote clinical research From past to future, the far-sighted men attached and will continue to attach importance to the development of animal models of human gliomas Judging from the research and development process, human beings have, after a hundred years’ efforts, moved from the development and application of animal models of spontaneous and induced brain tumor to the generation of the experimental platform of various animal models of human brain tumors Now we are trying to improve the simulation of the animal models to the human diseases For the human glioma, the early model was the solid tumors formed by directly inoculating in vitro passaged cell lines to animals; then, human glioma tissue were successfully inoculated into animals In recent years, with the progress of tumor molecular biology, transgenic or gene knockout procedures are used to generate genetic engineering mouse brain tumor model, which meet the requirement of finding the molecular etiology of human brain tumors through specific molecular genetics After the successful cloning of glioma stem cells, the establishment of animal models retaining the characteristics of glioma stem cells is on the agenda In short, although we face difficulty building up animal models

of brain tumor, we have tried to imitate the models to diseases from the system-cell level to the system-cells -molecular level

2 The types of tumor model

2.1 Allogeneic graft model of mouse brain tumor

Although allogeneic grafting of animals’ spontaneous tumors succeeded before, this method had been abandoned because of its poor simulation such as the low incidence, the early stage occult and short survival period of tumor-bearing animals It has been replaced by the development of induced animal brain tumor model The most commonly used cancer-

inducing methods are by chemical carcinogen or by viruses Back to 1939, Seligman et al reported that implantation the pill made of the polycyclic aromatic hydrocarbons methylcholanthrene in mice brain induced glioma and sarcoma From the middle of last century, the systemic administration of pro-nerve alkylating agent was proved to induce nervous system tumors Since then, the polycyclic aromatic hydrocarbons-induced method has been gradually replaced by nitrosourea derivatives-induced methods, especially by the methods using nitrosourea and ethyl nitrosourea which have higher rates of inducing central nervous system tumors Nitrosourea can induce tumor effectively in adult mice It can induce astrocytoma, oligodendrocytes, ependymal tumor or the most commonly glioma, which is the mixture of all the previous tumor cell types The intravenous injection

of ethyl nitrosourea to the 20 days pregnant rats induced the central nervous system tumors

in all the offsprings The widely used P494, C6, 9L and G422 animal glioma models are all produced by the similar methods The sensitivity to carcinogen of rat central nervous system

is formed 10 days before birth, and reaches the peak at birth (50 times sensitize than the adult) One month after birth, the sensitivity drops to the adult level

In addition to the chemical carcinogen, oncogenic virus is also used to induce gliomas The virus can induce two types of brain tumors: RNA viruses, such as Rous sarcoma virus or DNA viruses, such as adenovirus Concentrated Rous sarcoma virus (0.01ml) have been injected into the brains of newborn dogs, which all had gliomas after a period of latency (Bigner et al, 1970) It was also reported that direct injection of AD12 virus into the brain of mouse that had been born for 24 hours induced brain tumors after an incubation period of several months, most of which are medulloblastoma Usually for the newborn rats, tumor is formed 9-100 days after birth, the longest one takes about a year to mature after viral transfection Tumor cells were implanted by intracerebral inoculation of 4 X 107 chick embryo fibroblasts infected with the Schmidt-Ruppin strain of Rous sarcoma virus (RSV) With a 15 to 67 day latency, brain tumors were induced in 11 (73.3%) of 15 RSV-inoculated monkeys (Tabuchi et al, 1985) Scientist also found that inoculating the viruses isolated from brain tissue of progressive multifocal leukoencephalopathy (PML) patients into hamsters’ or monkeys’ brains could induce cerebellum medulloblastoma, hypothalamic gliomas, pinealomas, intraventricular ependymoma and many other types of brain tumors in various locations Brain tumors caused by viruses can only be produced into models with stable biological characteristics by cloning, such as the RT2 glioma model induced by the chicken tumor virus

These chemical carcinogens and oncogenic viruses are prevalent in the human environment, which which imitates the natural occurrence of spontaneous human gliomas The induced tumors in animals will be continuously passed through generations and the biological characteristics of tumors are relatively stable, which play active roles in understanding the tumor development and in preventing tumors However, animal models of brain tumors induced by oncogenic have different cycles and different pathological types Compared to human brain tumors, the induced brain tumors in animals were different in genetics, cell biology and histology Researchers hope to develop the xenograft model of human brain tumor to improve the simulation

2.2 Xenograft model of human brain tumor

2.2.1 Animal strains

Typically, it is impossible for the human tumors to grow in the animal due to strong immune rejection So the tumor is usually inoculated in the anterior chamber or parts of the hamster

cheek pouch where immune cells can not reach But these tumors have very unstable biological characteristics such as spontaneous regression, so the above method is only used for tumorigenicity testing We have injected immunosuppressive agents dexamethasone in newborn rats to successfully inoculate the human glioma cell line SHG44 into the brain of Westar rats, which is the first experiment to use human glioma cells for in situ animal tumorigenicity experiments Later, due to the appearance and wide usage of immune-deficient animals, the injection methods of immunosuppressive agents have been abandoned

Internationally, there are more than 30 kinds of pure T cell deficient nude mice with clear genetic background available, as well as T cells and B cells combined deficient mice (Lasat), SCID (severe combined immunodeficiency), NOD-SCID, CBA / I mice, and Beige mice with T cells and NK cells double deficiencies The nude mice currently used in China -

- Balb / C, Swiss, and NC strains were imported from abroad in the early 80s of last century The NC strain mice introduced from Japan in 1981 are non-inbred nude mice with high reproductive rate; they are resistant to pathogens and easy to manage They are still used for establishing human glioma models NHG-1

The recent established green fluorescent protein (GFP) transgenic mice C57BL/6J-GFP are very popular because it is easy to trace the green fluorescence in the host tissue or cells of tumor xenograft models However, tumor xenografts could not be established because of their normal immune function To apply them in human cancer model transplantation, Yang and colleagues (Yang et al, 2004) successful hybridized them with nude mice to produce immunocompromised nude mice expressing GFP which are suitable for human cancer transplantation We have also successfully cultured the NC nude mice expressing GFP (Figure 1), and these mice have been used in human glioma xenograft experiment

2.2.2 Method of transplantation

Commonly, tumor cell lines cultured in vitro, tumor tissue or the cell suspension digested from tumor tissue are used for eatablish xenograft model Usually, the implantation sites can be subcutaneous space, foot, abdomen, renal capsule, intracranial brain parenchyma, ventricles or spinal subarachnoid space, depending on the experimental need In the early stage, we established the NHG-1 solid tumor subcutaneously in nude mice model using the human brain malignant astrocytic tumor cell lines implantation, the NHE-2 nude mice xenograft model using human ependymoblastoma tissue implantation and the mouse - human chimeric immune mice model of human glioma using human peripheral blood mononuclear cells SCID transfusion By subcutaneous xenograft, it is not only convenient to observe the tumor volume by visual or dynamic measurement, but also easy to evaluate the effects of anticancer drugs However, the tumor formed in this way is not in the brain and the blood-brain barrier, the macro- and micro- environment of tumor cells are quite different from those in clinical diseases Therefore, the orthotopic transplantation model of human brain glioma in nude mice is a better model for imitating the clinical diseases

The animal model of glioma orthotopic transplantation used in previous researches usually applied cell suspension cranial injection or tissue inoculation with craniotomy (Antunes L et

al, 2000, Bradley NJ et al, 1978, DeArmond SJ et al, 1994, Horten BC et al, 1981, Rana MW et

al, 1977, Taillandier L et al, 2003) The former method can be used to generate the tumor model, but the procedure is too complicated There are lots of issues, for example: (1) the tumor cells for inoculation are usually cultured in vitro for several generations, which are damaged during the trypsin digestion It is difficult to get enough living cells; (2) the injection volume and speed are restricted by automatic pump because of the small

compensatory volume It will take a long time to make the model; (3) the inoculation cells are out of the incubator for too long to keep all alive because the operation takes too much time Although the same amount of cells was used in different batches of xenograft, it is hard to get about the same number of alive cells in every experiment, which have impact on the tumor-inducing rate and latency Although the later method can avoid the above problems, there are still lots of concerns such as large craniotomy injury to mice, complicated operation and other issues We used needle for transplanting tumor tissue in either subcutaneous or intracerebral space, as shown in Figure 2 In such way, the trauma was relatively small Compared with the cell suspension, tissue transplantation inoculated suitable environment (stroma) at the same time It is better in maintaining the original parental tumor structure, tumor biology or molecular phenotype

Fig 1 The proliferation and fluorescent protein expression in nude mice transfected GFP: A row, IVC system, an independent air supply cage for mice, produced in Suzhou, consists of

4 parts: the air supply system, exhaust system, cage, mouse box Fan is imported from German; high efficiency filter is produced by Aetna, a chinese-Japanese jointed venture; differential pressure gauge is imported from the United States The cage is made of

imported stainless steel tubes 304 The rat box is made of polysulfone transparent material

B row, from left to right are NC male mice used for breeding, neonatal of GFP/C57 female mice, bred GFP/C57/NC nude mice and adult mice C row, from left to right are

GFP/C57/NC nude mice under anatomy, eye view of mice brains, the cerebral hemispheres and bone marrow biopsy under fluorescence microscopy

A

C

B

Fig 2 The diagram of intracerebral and subcutaneous tumor xenograft transplantation: A-K: the surgical instruments, including micro-cranial drill (A), a metal trocar for subcutaneous inoculation (B), plastic trocars (C, D) and plastic trocars for intracerebral inoculation (E, F) For intracerebral inoculation, an external pin 2mm or 3mm shorter than the inner sleeve of the jacket tube (F, white arrow) must be used to control the depth of the puncture (if

necessary, use the caliper G for measurement) Use the forceps to move the tumor tissue (I)

on the top of the trocar (J, arrow), then use propeller (H) to push the tumor tissue into the casing, any extra tissue will overflow automatically from the needle end, and then place the core needle, push ahead to the second stent level (2-3mm length of tumor tissue is still left in the casing, at this piont, K) In intracranial inoculation, the anesthetized animals should have scalp incision, and drilled at 2.5mm right to the sagittal suture, 1.0mm to the cranial coronal suture (L), the needle to be vertically inserted into the brain (2mm or 3mm) and the inner core is pushed slowly until the tumor tissue be removed from the casing into the caudate nucleus (M, N arrow) If the tissue need to be inoculated subcutaneously in the puncture site (usually in the right armpit), use ordinary needle to puncture a hole, and then put the metal casing filled with tumor tissue (P) in the puncture holes, push the core of the casing to move the tumor tissue into the subcutaneous (O), slowly remove the needle Plastic trocar also can

be used for inoculation (Q)

There are essential indicators for evaluating the inoculation quality: transplantation success rate and the stability of parent tumor characteristics According to the author's experience, the success rate of brain tumor xenografts is determined by the following factors (1) For the subcutaneous inoculation, the nearer the location is to the head, the easier it is to generate the model (2) Compared with using human tumor tissue, it is easy to generate the model by

using the established tumor cell lines The in vitro cultured tumor cell lines have stable

biological characteristics and proliferate very fast, which make it ideal for establishing xenograft model The tumor tissue inoculation keeps the original tumor characteristics in simulating the blood supply, interstitial structure and growth characteristics (3) It is more successful to use tumor tissue inoculation than using trypsin digested tumor cells inoculation The latter method can provide the exact amount of inoculation cells (4) The more cells inoculated, the shorter the incubation period would be (no less than 1 × 107 cells

in the initial inoculation) But Singh's group (Singh et al, 2003, 2004) has reported that it was the number of tumorigenic stem cells, rather than the number of total cells, that has decisive effect The study also showed that 102 tumorigenic stem cells are sufficient for tumor generation, while 105 non-tumorigenic stem cells would not (5) Compared with inoculating

in subcutaneous space and in the abdominal cavity, it is easier to generate the model by inoculating in the peri-renal adipose capsule with relatively low immunity or in intracerebral space

To determine the stability of transplantation tumor biologic characteristics, the models must

be evaluated by the following indicators: (1) maintain the genetic characteristics: the chromosome of transplanted tissue is same as the primary inoculated human tumor tissues; (2) maintain the morphological features: the morphology, mitotic status, tumor stroma and vascular structures of transplanted tissue are identical to the primary inoculated human tumor tissues under the light and electron microscopy; (3) maintain the tumor markers: the specific qualitative biochemical indicators or quantitative biochemical indicators of transplanted tumor tissues are consistent with those of the primary inoculated human tumor tissues; (4) maintain the stability of the biological characteristics of tumor-bearing animals: after several generations, when the transplant success rate reaches 100%, there are less difference in spontaneous regression, tumor size and the survival rate between tumor-bearing animals; (5) maintain the proliferation kinetics of tumor cells: the mitotic index, phase, cell cycle and doubling time are almost the same According to the above criteria, we have successfully established a primary malignant glioma and lung cancer brain metastases orthotopic transplantation nude mice models

With the progress in the cancer stem cell research, it seems the quality control of animal models should be focused on the probability of replicating the parent tumor stemness characteristics As is mentioned above (Singh et al, 2003, 2004), Singh’s group found that there are inconsistent cellular renewal and proliferation rate in human brain tumors They have isolated CD133+ tumor stem cell, the only subtype that can cause intracranial tumors, and proved that CD133+ cells are the tumorgenic cells in human brain These findings make the generation of stem cell-induced brain tumor animal model possible XioNan Li's group had generated the real animal models using human brain stem cells (Shu et al, 2008) 17 cases of pediatric glioma specimens were grounded into single cells or 3-5 cells suspension, and were injected into the NOD-SCID mice brain or cerebellum using the needle He built 10 animal models for human glioma xenograft Evaluated by HE staining and the critical markers, especially CD133 immunohistochemistry, the transplanted tumor retained the parental tumor characteristics, like invasion, histological type and BTSCs and stem cell pool expressing CD133 + In the tumor neovascular, the human specific CD31 and CD34 were negative, while the human /mouse shared vWF was positive The results indicated that the tumor neovascular was provided by the host It is controversial, because previous report indicated that BTSCs provide the neovascular BTSCs originated angiogenesis have been

found in vitro and in vivo Our results were different from those of Li’ team, because we used

tissue inoculation while Li et al used single-cell or 3-5 cells cluster inoculation; these two methods may provide different micro-environment for BTSCs We think that tissue transplantation will better maintain the characteristics of origin tumor

2.3 Genetic engineering model of mouse brain tumor

In 1984, Brinster et al fused the promoter (MK) with polyomavirus SV40 gene After enzyme digestion, he microinjected the DNA fragments into male pronucleus of fertilized eggs to built transgenic mice The transgenic mice developed a variety of tumors, such as choroid plexus papilloma, thymoma and tumors in endocrine system In 1988, Reynold et al used H-2kb/ SV40 infusion to generate the transgenic mice, which provided results similar to Bringster’s Later, Vogel et al fused HTIV-LTR with tat gene, the injected transgenic mice can spontaneously develop neurofibroma In 1990s, the transgenic glioma model was finally generated Studies showed that there were lots of similarities between the making of glioma model and other tumor models Some important DNA elements, such as the promoters and enhancers, modulated the transfected gene; the foreign genes fused with promoter and enhancer promoted a strong expression of target genes The promoter or enhancer can be located in the upper stream or down stream of the genome, or it can be inserted in the non-transcribed DNA fragments (introns) There are constructed transgenic mice with astrocytoma, medulloblastoma, oligodendroglioma and multiforme glioblastoma

2.3.1 Genetic engineering mouse of astrocytoma

Bachoo et al (Bachoo et al, 2002, Xiao et al, 2002) developed the mouse glioma model using retroviral carrying the active EGFR to infect astrocytes They confirmed that injection of Ink4a/Arf-/- astrocytes expressing EGFR formed spheres into the animal's brain induced the formation of the high invasive astrocytoma To simulate the RB mutations, Terry Van Dyke et al generated mouse astrocytoma model by introducing SV40T antigen (T121) which bind with RB and RB family members, P107 and P130 under the control of glial fibrillary acidic protein (GFAP) promoter In 1995, Andrew Danks et al reported GFAP/SV40 TAg transgenic low-grade astrocytoma model GFAP is the primary marker to identify the origin

of the tumor, while SV40 TAg binds to P53 and inhibits its activity After these two proteins were combined, the former regulated SV40 TAg and promoted its development of astrocytic tumors, while the latter suppressed the activity of wild-type p53 and promoted tumor development As was shown in morphology and immunohistochemistry, astrocytes mainly were located in the subependymal zone in GFAP/SV40 TAg transgenic mice Consistent with the characteristics of astrocytomas, some were located in the brain parenchyma or around neurons and blood vessel with GFAP expression Chinese researcher Li HD reported that transfection myc andSV40TAg would promote cerebellum neuroblastoma and pancreatic cancer development

2.3.2 Genetic engineering mouse of oligodendroglioma

Myelin basic protein (MBP) is an over-expressed protein during the formation of nerve myelin, which located in oligodendrocytes in the central nervous system or in Schwann cells

in the peripheral nerves system Oncogene neu was highly expressed in the majority of gliomas In 1992, Hayes recombined Neu gene isolated from PSV2neu NT plasmid with MBP isolated from Cosmid (cos138) clones, and implanted it into C57BL / 6 × DBA / 2 F2 fertilized eggs Of the 93 fertilized eggs injected with the recombined DNA, 14 of them had offspring Four of the neonatal had brain tumors, which appeared in the underside of the

brain, the thalamus and the posterior fossa and compressed the brain stem Under the light microscope, the brain tumors of three mice had the characteristic morphology of glioblastoma multiforme, and had extensive leptomeningeal invasion Under the electron microscope, there were some characteristics of undifferentiated cells To identify the origin

of tumor cells, they used NF, GFAP, MBP and Leu 7 immunohistochemical staining The results showed that there were GFAP, MBP and Leu 7 positive staining in relatively well-differentiated tumor cells, while in low differentiated tumor cells, less GFAP, MBP, and LEU

7 positive staining were found None of the cells had NF positive staining For the morphological analysis, Hayes's experiment did not successfully induce typical oligodendrocyte tumor, although the expression of MBP and Leu 7 indicated the presence of oligodendrocytes The hybridization analysis was further used to clarify its origin, the results showed that a large number of neu gene presented in tumor cells, but not in normal brain cells In addition, the results also showed that RNA level of myelin specific proteins MBP, PLP, MAG and CNP were 5-10 times higher in tumor cells than in the control group, which suggested that the myelin-forming cells was enriched in the tumor There was no myelination protein Po found in peripheral nerve, indicating that tumor cells were derived from oligodendrocytes, but not from Schwann cells Moreover, there was no NF detected in brain tumor cells, indicating that there was no neuron present in the tumors Analyzed by the solid tumor markers, Hayes had established the transgenic mice of oligodendrocyte cell tumors

2.3.3 Genetic engineering mouse of glioblastoma

Injection of the combination of activated Ras and AKT into an Ntv-a transgenic mice induced glioblastoma tumor (Holland et al, 2000) When the DNA mixture was injected into Ntv-a mice with inactivate NK4a-ARF, the formation of the glioblastoma was accelerated Ras and Akt work on the downstream signaling pathways of several growth factor receptors In most of the glioblastoma multiforme tumors (GBMs), Ras and Akt are activated simultaneously It has been proved that the abnormal expression of Ras can inhibit p53/RB pathway which induced the transformation of astrocytes into anaplastic astrocytoma cells that finally obtained GBMs characteristics after transfection AKT in the cells These results confirmed the combined action of Ras and AKT in malignant glioma Another GBMs model

is generated in Nf1 and p53 double-silent mouse mated by the p53 knockout mice and the heterozygous of Nf1 and Cis (Reilly et al, 2000) Transplantation cells from such mice into other mice induced glioma or GBMs, which was caused by a depletion of a tumor suppressor gene instead of overexpression oncogene Despite the molecular differences in previous GBMs models, they have something in common Nf1 (nerve fiber) suppresses Ras activity, Nf1 knockout would lead to RAS activation Nf1 alone is not sufficient to induce tumor formation, Nf1 combined with p53 deletion or lack of cell cycle regulation will induce tumor formation

2.3.4 Genetic engineering mouse of medulloblastoma

Generation of Ptch heterozygous mice is essential for the development of medulloblastoma transgenic mouse model It is well known that Ptch receptor suppress proliferation through SHH / GLI signaling pathway Inactivated Ptch receptor increases the risk for medulloblastoma, about 14% to 19% of the Ptch +/- mice develop medulloblastoma within

12 months, indicating that retained Ptch locus still function When these mice were mated with the p53 deficient mice, the tumor incidence of their offspring increase to 95%, all

affected mice die within 12 weeks (Zurawel et al, 2000, Wetmore, 2001), suggesting that P53 plays an important role in this model

The systemic experiment on the retroviral model and RCAS/tv-a indicated that SHH pathway was related to the formation of medulloblastoma Under the guidance of ultrasound, utero injection of retroviral SHH directly into the cerebellum could induce medulloblastoma (Weiner et al, 2002) Fults et al over-expressed SHH in newborn mouse cerebellum using RCAS / tv-a system, which induced the formation of medulloblastoma c-Myc over-expression enhanced the induction SHH's activities require the participation of Ptch Inactivation of p53 and RB genes in neural progenitor cells in cerebellar granule cell layer developed brain tumor, indicating that RB family proteins may work on the tumorgenesis P53 and RB conditional knockout mouse can produce a medulloblastoma, while single inactivated p53 or RB genes has no such effect Some studies have shown (Tong

et al, 2001) that adenosine diphosphate ribose polymerase (ADRP) is an early DNA damage response molecule The mouse lack of ADP-ribose polymerase mate with the p53 gene deficient mice to generate ADRP and p53 double deletion mouse Half of the mice have medulloblastoma located in the cerebellum

3 Pathological features of transplantation tumor

When human cancer tissue or cells are transplanted into animals, the micro-ecological environment has undergone tremendous changes The cell morphology, molecular biology, host survival period, clinical symptoms of tumor are difficult to keep consistent with those

of original tumors in many ways Tumors developed with single cell suspension or ectopic transplantation have the interstitial and vascular components provided by the host (mouse), which is significantly different from clinical disease For example, in human glioma subcutaneous xenografts model, the tumor weight up to 5-6 grams is not life-threatening to the 25 grams weighted nude mice For the intracranial tumor, even when the inoculated tumors cover the entire cerebral hemisphere, or grow into the contralateral hemisphere, the cranial tumor-bearing mice are still alive (Figure 3) Therefore, to study the pathology of transplant tumor, we have to link them with clinical as closely as possible There is no much references available, so we shared a few research data with all the readers here

3.1 Homogeneity

Xenografts inoculated in nude mice can induce the transplant tumor having pathological features similar to the clinical specimens Usually, the subcutaneous xenografts in nude mice are different from the original tumor Brain glioblastoma multiforme tumor (GBM) transplanted subcutaneously in nude mice does not have the typical GBM features, but more like "fibrosarcoma" When the xenograft is inoculated in the brain, some invasive characteristics and molecular markers are consistent with the parental tumor Our data proved that the parental tumor is non-invasive and overexpression of CEA, with acidic mucus secretion, the brain tumor metastases from lung cancer in nude mice can mimic these characteristics While the highly invasive GBM with high expression of EGFR inoculated into nude mice brain showed highly invasive and EGFR over-expression characteristics For morphology analysis, it is rarely identical The morphology of implanted tumor has different characteristics depending on different locations, such as cerebral cortex, white matter, gray matter, ventricles and cerebellum

Fig 3 NC glioma in nude mice, SCID mice transplanted subcutaneously, dexamethasone immunosuppressed Wistar rats and brain transplantation model in Balbc nude mice: the cells in subcutaneous space and in brain grow into large tumors due to unlimited

proliferation Intracranial skull uplifting can be observed in some mice (C, D black circle) Observed with cranial dissecting microscope or bear eye, the transplanted tumors almost covered the entire hemisphere (C arrow), or reached the olfactory lobe and the contralateral frontal lobe (D arrow) If spread to the choroid plexus, the tumor will cause hydrocephalus, both hemispheres are highly swollen (E) and ventricular expansion (F) will be noticed

3.2 Mimesis

In the tissue repair process involving neural stem cells, the amplified daughter cells must have the same type of repair tissue to rebuild the function of cells, a phenomenon known as mimesis of neural stem cells, which had already been confirmed We only focus on what the xenograft grows like and the tissue types of the parental tumor When the glioma stem cells were implanted, we only checked whether the xenograft had neurons and glial cells features, rather than clarifying the subtype of the tissue Our experiments have shown that stem cells derived from GBM grew diffusely, when it is located in the choroid plexus, it developed into choroid plexus carcinoma (Jun et al, 2010); when it settled in the ventricle wall, it developed into the ependymal neuroblastoma; when it settled in the brain surface

and spinal table, it developed into uniform small round cells (do not know the subtype) In short, stem cells derived from GBM implantation has no typical GBM characteristics, the type of transplant tumor highly depends on the location We believe that glioma stem cells, like neural stem cells, have mimesis growing characteristics

3.3 Remodeling

The tissue remodeling during the development is very common In tumor development and progression, it is not conclusive whether there is remodeling between tumor and the host tissue cells, such as interstitial and vascular Tumor vessel remodeling was first discovered

in the malignant retina melanoma and the xenograft (Maniotis et al, 1999, Zhang et al, 2006) The vessels composed of tumor cells are called vessel memisis; while the vessels composed

of tumor cells and endothelial cells are called mosaic blood vessels We have made detailed observation on tumor vessel in malignant glioma (Yue et al, 2005) In human glioma transplanting into nude mice, the glioma stem cells migrated and proliferated around the host blood vessels Besides relying on the host for the blood supplies, the tumor had spontaneous vascular memisis and mosaic blood vessels to provide nutrition for rapidly proliferating tumor Moreover, some vascular wall cells had both host protein and tumor protein expression, indicating that in the process of tumor angiogenesis, the tumor stem cells as well as tumor-derived endothelial cells were directly involved

There are several types of cancer stem cells and host cells remodeling Besides the vascular remodeling, it is possible that the individual suspension glioma stem cells could remodel with host cells There are following possibilities: (1) individual tumor cell relies on the host stromal cells for nutrition supply, therefore remodeling with the host vessel; (2) individual tumor cells fuse with host cells or other tumor cells to form multinucleated giant cells or aneuploid cells, which use the fusion cells to rebuild tumor blood vessels; (3) in very few cases, individual tumor cells, in particular disseminated tumor cells, transdifferentiate into vascular endothelial cells and build stem cell niche (Niche), which expand to form a distal distribution of tumor cells block With the tumor progression, the host tissue gradually disappears among tumor mass

3.4 Tumor imaging

The earlier imaging study about the animals bearing human brain tumor used single photon emission computed tomography (PET) Using tumor monoclonal antibody labeled with iodine radionuclides, the biological imaging of subcutaneous tumor had been collected by the γ camera and SPECT (single photon emission computer-aided tomography) machine in tumor-bearing mice, which may guide the diagnosis (Haubner et al, 2001, Herschman et al,

2003 Massoud & Gambhir, 2003,) 18F Radionuclide labeled glucose associated with tumor cell metabolism is also been used to trace tumor proliferation activity Since most of the tumor-bearing animals are rats or mice, inoculated brain tumors are difficult to distinguish, both methods only work for the subcutaneous tumors With the improvement in scientific technology, foreign researchers have used animal-specific high magnetic field MR (3-17.6T)

on rats or mice for conducting imaging studies of intracranial tumors (Beck et al, 2002, Lewis

et al, 2002, Pirko et al, 2005) Chinese researchers are still using ordinary field MR (1.5T) for imaging studies of rat intracranial tumors Recently, we used rats and mice specific small coil 1.5T MR machine for imaging studies of orthotopic glioma in nude mice We got some satisfactory results, which can be used for monitoring tumor and adjacent structures and

calculating tumor volume But it will take a long imaging time and can not mimic the patient pictures

Tumor vessel imaging is part of the tumor imaging Percutaneous transcatheter and intravascular injection of contrast agents, and digital subtraction angiography (DSA) are used to get tumor microvascular data However, the transplanted tumors mostly use small rodent animals, on which the DSA technology can not be applied The vascular endothelial cell marker, such as CD34 + was used to calculate the vascular density and evaluate the number of tumor blood vessels, and the therapeutic effect of anti-tumor angiogenesis drugs However, this method can not be used to evaluate the transporting function of these vessels The cancer stem cell self-generated blood vessels (vasculogenesis) different from the host vascular endothelial cells formed blood vessels (angiogenesis) We established the activated carbon granular heart chamber perfusion method, which confirmed that various types of tumor vessels are involved in tumor-bearing animal systemic circulation (Figure 4) (Dong et

al, 2010) The detailed procedures are as following: (1) producing activated carbon suspension: activated carbon particles were ground into powder and added to the PBS to make the suspension Then the mixture was filtered through 40-micron net (U.S BD Company) After rested for 1 min, the carbon particle was sucked by a flat cut, polished needle; (2) infusing activated carbon suspension: the tumor-bearing mice were anesthetized

by 10% chloral hydrate The syringe was pierced through the left ventricle of mice, while a small hole was cut in the right atrial appendage of the heart to facilitate the replacement of circulating blood After 2-3 ml of carbon suspension was infused, the tumor tissue was removed, fixed in 4% paraformaldehyde and embedded in paraffin; (3) analyzing the perfusion results: active carbon particle suspension in left ventricular cavity went into host circulation and the tumor blood circulation The active carbon particle distribution was related to the blood density There were small carbon particles in host large vessels and new bleeding necrotic area, and integrated condensate active carbon particles in the medium-sized blood vessels, aggregated carbon particles in the tumor microvascular It is worthy to mention that the active carbon particles would leak out of the lumen because the carbon particle is less than 40 microns in diameter, smaller than the red blood cells It is interesting that the carbon particles were present in the marker of highly malignant tumor "false daisy group" and "tumor necrosis" For the former one, few carbon particles were observed in the central lumen, while in the latter structure, large piece of carbon particles were scattered in the gap of sparse distributed tumor cells Since there is no red blood cells or other tangible materials found, suggesting that this change existed after tumor necrosis The present carbon particles indicated that there was nutrition supply, suggesting that necrotic tumors tissue might have a micro-environment promoting "self-healing" process

Imaging of fluorescent protein tracer is a newly developed technology We had inoculated human glioma stem cells SU3 transfected with red fluorescent protein (RFP) into BAlbc nude mice brains to trace its location (Fig 5) Hoffman (Hoffman, 2002) had inoculated U87 glioma cells transfected with RFP into the brains of nude mice expressing green fluorescent protein (GFP) Farin et al (Farin et al, 2006) had injected C6 glioma cells labeled with eGFP and DsRed-2 into the forebrains of neonatal rats, and used fluorescence imaging to observe

dynamic tumor growth in vivo Although clarity of the imaging, precision of the display of

tumor size, location and depth are still not satisfying, this method will be widely used with the development of technology In this platform, the outstanding finding was that glioma cells invaded brain tissue along the cavity of blood vessels Tumor cell intruded into the endothelial cells and the pseudo foot of astrocytes, not the vascular cavity In addition, glioma cells jumped forward sometimes slowly and sometimes rapidly, with the maximum

Fig 4 Carbon particles ventricular perfusion in tumor-bearing mice, to trace blood flow of new Integrated tumor vessels When carbon particles are perfused in tumor-bearing mice hearts, the blood flow of new tumor vessels is showed as following: A: suspension of carbon particles by light microscopy; B: the ventral surface profile of brain tumor, showing the basilar artery and Willis ring filled with carbon particles; C-K: the HE sections, showing that the carbon particles go into the following vascular tissue: subarachnoid space (C); choroid plexus (D); normal brain tissue (E); tumor margin (F); tumor foci (G); massive tumor tissue, including some endothelial-dependent blood vessels (H, host blood vessels) and some blood vessels formed by tumor cells (I, vascular mimesis); carbon particles leaking from tumor-origin blood vessels (J, arrow in magnified square); carbon particles in the central vessel of false daisy (K, circle); besides, carbon particles existing outside of vessels in the tumor necrosis, in acute necrotizing period, carbon particles and floating red blood cells coexist (M); after the acute period, carbon particles exist in absorbed lesions (N), indicating that there are blood supply even in the repair period of tumor necrosis

speed over 100um per second; the migrated cells would go through proliferation and division Cells divided at or near the vascular bifurcation This was the first time the comprehensive kinetic data about glioma cell infiltration in vivo was recorded, which indicated that the proliferation and migration of glioma cells related closely with host vascular system Considering from the reconstruction of tumor tissue, host's own tissues and cells also play an important role and provide nutrition for the growth of tumor Yang (Yang et al, 2004) and we had inoculated human glioma stem cells non-transfected and

transfected with RFP into nude mice brains The former model (Jun et al, 2010) had proved that cancer stem cells involved in tumor blood vessel formation and fusion with host cells;

In the latter model, host cells were proliferating actively around the tumor, and active host cells cultured in vitro have immortalized features (Figure 6)

Fig 5 Tracing human glioma stem cells SU3 and orthotopic transplantation tumor in Balbc nude mice with red fluorescent protein (RFP)mice: A and B: the SU3 with RFP transfection cultured with growth factors or serum under optical phase contrast microscope; C and D: the brain tumor observed in natural light and excitation light, red swollen tumor in th front

of the brain; E and F: the tumor tissue sections under confocal microscope, the red tumor cells (E) and the nuclear staining (F) crowdedly arranged, suggesting a high degree of malignancy

Fig 6 The image of RFP / GFP brain tumor biopsy under fluorescence scanning confocal microscopy RFP/GFP brain tumor biopsy under fluorescence scanning confocal microscope (A), human glioma stem cells (SU3) transfectded with RFP is red, host (NC/C57BL/6J-GFP nude mice transfected with GFP) tissues and cells is green, the nucleus is blue There are host tissues in tumor interstitial and around the active zone, where the high proliferative host cells crowed together The morphology are diverse among the cell suspension cultured from the tissue taken from the active zone under the the fluorescent microscope Some of them are pleomorphic tumor cells, with several features: star shape, highly proliferative, rapidly covering the culture bottom, monoclonal passaged (B); others are macrophages with putiplnucleus (C); neurons (D) and fiberblast (E)

The establishment of fluorescent protein transfected tumor cells and transgenic mouse tumor model has made a significant contribution to the medical research There are lots of application followed by pitfalls: (1) although fluorescent protein is less toxic to cells, it has cytotoxic effects to liver, heart and nervous stem cells (Huang et al, 2000]; (2) tumor cells expressing fluorescent protein may be engulfed by phagocytic cells invaded into the tumor area, it is impossible to distinguish them using current technology, which may lead to a false judgment of the experimental results; (3) limited by the current technology, the method can not fully meet the needs of interpreting fluorescent tracer image However, we have every reason to believe that with the introduction of new imaging technologies, fluorescent tractor will make greater contribution to cancer research For example, neural stem cells and cancer stem cells transfected with different colors of fluorescent protein are likely to help distinguish the origin of tumor cells; in the tumor micro-ecology research, the improved

technology will help to dissect the components of cancer stem cell niche; in tumor evolution,

it is known that anaplastic cell is the basis for malignant tumor, tumor cells and host cells transfected with different colors will help to elucidate whether cell fusion could contribute

to the malignant progression Finally, during the reconstruction process of transplant tumor, the donor and receptor with different fluorescent proteins are expected to be used to record their roles non-invasively

4 Acknowledgments

This chapter is supported by National Basic Research Program of China (973 program, 2010CB529403), National Natural Science Foundation of China (No 30872654, 81071766), Natural Science Foundation of Jiangsu Province (NO BK2008173, 2010227) and the Foundation of Suzhou YJ S0950

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Experimental Brain Tumors: Current Concepts

Jesús Vaquero and Mercedes Zurita

Neuroscience Research Unit, Hospital Puerta de Hierro-Majadahonda, Madrid

Spain

1 Introduction

The possibility of having experimental models of brain tumors allows for testing therapies applicable to human brain tumors They can be induced by viruses, chemicals or radiation Radiation-induced brain tumors have seldom been used, but diverse virus groups have been used to induce brain tumors Among DNA viruses, both adenoviruses and papovaviruses have been shown to induce brain tumors in animals The RNA viruses causing experimental brain tumors have consistently belonged to the retrovirus group, and have been generally limited to the murine sarcoma virus, the avian sarcoma virus, and the murine sarcoma virus In these models, brain tumors are induced in rodents after intracerebral inoculation, with a variable latency, and the induced tumors are generally classified as gliomas, sarcomas, ependymomas or choroid plexus tumors

On the other hand, the heterotransplantation of human brain tumors into immunodeprived animals gained great interest after the development of the nude mouse model, a thymus-deficient animal that provided the possibility for the xenografting of human brain tumors It is known that human meningiomas and glioblastomas can grow after subcutaneous transplantation into the nude mouse, maintaining its original morphology Nevertheless, at present, diverse chemical agents provide the best models of experimental neurocarcinogenesis

2 Viral neurocarcinogenesis

The role of viruses in human oncology is a question that has interested for many years to researchers and clinicians (Bigner and Pegram, 1976) However, despite the intense research that has been developed over the last decades in this field, we still can not establish a clear etiological association between the presence of certain viruses and tumor development in humans, with some exceptions, such as the case of Epstein-Barr virus associated to Burkitt's lymphoma, although there has never been any conclusive proof that this virus causes the tumor

From an experimental point of view, one of the models used to trigger the development of neural tumors in experimental animals is inoculation by different routes of virus with oncogenic capacity (Bullard and Bigner, 1980) The potential value of virus-induced gliomas has been questioned, however the information obtained from these experimental models has enabled significant progress in the treatment of human cancers This experimental model of virus-induced neurocarcinogenesis offers the advantage that some of the viruses used will induce the development of tumors in a short period of time, the tumors are specifically located at the Central Nervous System (CNS) or Peripheral Nervous System (PNS), so that

we can not rule out the possible viral etiology of certain types of brain tumors in humans However, at present, numerous studies failed to establish any etiological association between viruses and humans brain tumors (Minn et al, 2002)

We now know different animal viruses that can act as transforming agents in normal cells, since they are capable of causing malignant transformation of a cell through its ability to integrate genetic information It is well known that, for example, intracerebral inoculation of retroviruses can induce brain tumors in a wide variety of animals Viral carcinogenesis allows us to induce experimental tumors with a short latency period and with a more specific location that offers radiation carcinogenesis, location depends on the route of administration, animal age and the amount of virus inoculated However, it is obvious that experimental models of viral neurocarcinogenesis have the inconvenience and risks involved in the handling of virus particles

Numerous studies have shown that RNA viruses (retroviruses) are able to induce the development of tumors in the CNS of experimental animals Within this group, we highlight the avian sarcoma virus, murine sarcoma virus and simian sarcoma virus, being the most widely used in experimental neuro-oncology Avian sarcoma virus (ASV) has been one of the most used in the literature to induce experimental brain tumors The tumors are usually induced in chickens by intracerebral inoculation, and intracerebral tumors originated showed characteristics of sarcomas There have been studies of ASV inoculation in the brains of monkeys because of their similarity to man, and tumors induced were fibrosarcomas Interestingly, no author has reported the existence of glial tumors, but it has shown the ability of this virus to infect and replicate in glial cells when they grow in tissue culture

Murine sarcoma virus (MSV) with its three strains: Moloney MSV, Kirsten and Harvey, can cause leukemia and sarcomas when inoculated subcutaneously in rodents and also is capable of inducing brain tumors in rats when inoculated intracerebrally Neoplasms that may result show usually the aspect of glioblastomas, gemistocytic astrocytomas, oligodendrogliomas and hemangioblastomas, depending on the age of the animal and the dose of virus inoculated

The simian sarcoma virus (SSV), after intracerebral inoculation in marmosets (Sanguinus nigricolli) induces the development of tumors that are morphologically similar to human glioblastoma multiforme, being able to demonstrate the presence of virus particles within tumor cells

Among the known DNA virus, adenovirus and papovavirus have proved very effective in inducing brain tumors after intracerebral inoculation in animals, preferably in neonatal age Intracerebral inoculation of polyoma virus induces a high incidence of intracranial sarcomas

in experimental animals, increasing their impact in terms of age of the animal (Rabson and Kirschstein, 1960) However, when inoculated cells transformed in vitro with the same virus, tumors of astrocytic aspect can be seen

The SV-40 virus shows no oncogenic effect in monkeys, a species from which it was originally obtained, but it is one of the more capable oncogenic virus in rodents Intracerebral inoculation in hamsters induces the development of ventricular tumors that were classified as ependymoma, choroid plexus papillomas and meningeal sarcomas The induction of brain tumors by this type of virus depends very heavily on the dose The role of SV-40 virus in human tumor development, not only brain tumors but also bone tumors and mesothelioma, has been subject of discussion for decades, but now there is conclusive evidence Furthermore, human adenoviruses can cause meningeal tumors when inoculated

intracerebrally into experimental animals, with tumor development after latent periods of 35

to 40 days

While most existing data in the literature refer to the oncogenic virus ASV virus and SV-40, other viruses whose first guest is the man also play an important role in viral neurocarcinogenesis, such as Ad12 virus, BKV and JCV, three DNA viruses that have been widely used in experimental studies designed to establish a possible relationship between virus inoculation and the development of brain tumors The human adenovirus Ad12 is able

to induce brain tumors in rats after intracerebral inoculation, with a greater susceptibility of neonatal animals, where the range of incidence may vary between 8 and 100% Furthermore, induced tumors develop after periods of latency between 31 and 235 days The human papovavirus BK is capable of inducing brain tumors with different histological features, when inoculated into experimental animals This virus is specifically used to induce choroid plexus papillomas and ependymomas after being inoculated intracerebrally

The JC virus (JCV), when inoculated subcutaneously or intraperitoneally into young hamsters, induces the development of a variety of tumors, especially mesenchymal neoplasms, and may even induce the development of peripheral neuroblastomas Moreover, intracerebral inoculation can induce the development of malignant astrocytomas In human neuro-oncology, this virus has been associated with the development of medulloblastomas and more recently, with recurrence of glioblastomas

On the other hand, it is important to note that in recent years, a new focus on the use of viruses has emerged in experimental neuro-oncology, and currently the use of viruses, or parts of them, are used as therapeutic vectors Although some success has been reached using oncolytic viruses in experimental treatments for malignant gliomas in humans, the fact is that so far the results with these new techniques do not appear to meet the initial expectations (Zemp et al, 2010)

3 Chemical neurocarcinogenesis

The discovery of chemical carcinogens has stimulated neuro-oncology research because, after systemic application, these compounds induce a high incidence of tumors in the CNS and PNS, such as demonstrated Druckrey et al in 1965, with the N-methyl-N-nitrosourea (MNU) Subsequently, we have found a greater number of chemical compounds, with equal effectiveness Some of these compounds only occasionally induce tumors in the CNS of adult animals, but they represent, however, powerful neuro-oncogenic agents when administered transplacentally or during the early stages of postnatal life Compounds, such

as propyl-nitrosourea, butyl-nitrosourea, dimethyl-nitrosourea, and Trimethyl-N-nitrosourea, have been used However, at present, N-ethyl-N-nitrosourea (ENU) is considered the best chemical agent to induce experimental brain tumors, because it

N-is capable of inducing a high incidence of tumors, with known latency and morphology

3.1 Mechanism of tumor induction in chemical neurocarcinogenesis

Most carcinogenic compounds actually represent precarcinogens which are converted in the host The final product of this transformation is an electrophilic group that is capable of reacting with various cell constituents It is clear that neuro-oncogenic compounds exhibit biological effects as alkylating metabolites which are formed during processing in vivo The molecular basis of malignant transformation is not fully clarified, and at present, the cell

being the target for the initiation of carcinogenesis has not identified Most investigations are based on the interaction of carcinogens with nucleic acids and proteins

Recent studies suggest the possibility that the induction of neural tumors by nitrosourea compounds may be related to a deficiency in DNA repair mechanisms in the Nervous System When applied 14C-ENU in neonatal rats, the loss of O6-ethylguanina in liver DNA

is very rapid However, it persists for several days in the cerebral DNA (Goth and Ralewsky, 1974) In the non-target organs for carcinogenic action, the O6-alkylation excision is repaired during replication, or alteration remains in the sequence of DNA bases

It is assumed that the inability of the neuro-oncogenic agents to induce neuronal tumors may be because neurons represent a cell population that has no capacity to divide The permanent genetic alterations are the result of a mutation (transition) and this requires DNA replication On the other hand, no direct evidence exists to affirm that the alkylation of nucleic acids is the cause that triggers the initiation of malignant tumors development

3.2 Factors affecting the induction of experimental brain tumors in chemical

neurocarcinogenesis

In chemical neurocarcinogenesis, the incidence, distribution, histology of tumors, and survival time of animals are influenced by the species and age of animals, in addition to dose and mode of application of the carcinogen

3.2.1 Species

The susceptibility of different species to the carcinogenic activity of nitrosoureas has been investigated by several authors Thus, Druckrey et al (1970) observed that strains of rats such as Sprague-Dawley and Fischer, Long-Evans and Wistar, were susceptible to the carcinogenic action, producing a high number of tumors in the CNS However, the response was not uniform, for example, male Sprague-Dawley rats treated with MNU only developed brain tumors, while male Fischer rats showed a high incidence of PNS tumors (Swenberg et

al 1972)

3.2.2 Age of animals

There is evidence that the response to neuro-oncogenic agents in fetuses and newborn rats differs significantly from the response in adult animals The main characteristics of the perinatal induction of tumors in the nervous system by chemical agents can be summarized

in the following points:

1 In adult animals, repeated doses of the carcinogen is needed to obtain a high incidence

of neurogenic tumors In the perinatal carcinogenesis, however, a single dose is sufficient to induce tumors in the nervous system, approximately in 90-100% of the experimental animals On the other hand, some compounds such as 1,2-dimethylhydrazine, only induce tumors in fetuses and newborn rats, but never produce neurogenic tumors in adults

2 Transplacental induction of neurogenic tumors in rats is possible only after day 11 of gestation This is not due to lack of penetration of the carcinogen in fetal tissue, because embryotoxic and teratogenic effects occur after treatment, during the early stages of development Nervous system susceptibility to chemical carcinogens increases sharply after day 11 of gestation and peaks during late intrauterine development period (Druckrey et al 1969) After the first month of postnatal development, the response to neuro-oncogenic agents is broadly similar to that obtained in adult animals

3 In adult animals, the tumors are located mainly in the brain (Denlinger et al, 1973) However, after perinatal application, tumors typically occur at the level of the spinal cord and the PNS Trigeminal nerve tumors occur more frequently when the carcinogen

is administered at the end of gestation, whereas in this case, the number of brain tumors

is less than when the carcinogen is administered on day 15 of intrauterine development (Kahle, 1970)

4 The prenatal administration of these compounds increases the neurospecific carcinogenic effect After transplacental administration tumors are located almost exclusively in the CNS Postnatal application also produces a significant number of extraneural tumors (Schreiber et al, 1972)

3.2.3 Dosage and application

The incidence and latency period of experimental tumors is highly influenced by the dose of carcinogen The number of tumors transplacentally induced by ENU can vary between 100% and 63% when the carcinogen dose is reduced from 80 mg / kg to 5 mg / kg Moreover, the latency period is increased from 180 days to 500 days, when the dose of ENU administered

in neonatal rats is reduced in the same way The mode of application of the carcinogen plays

a key role in the location and type of tumor that will be induced Thus, local application of nitrosoureas can induce the formation of local tumors, but when these compounds are administered by intravenous injection, they can produce tumors that are spread throughout the body

3.2.4 Hormonal and immunological factors

The possible influence of hormones on chemical carcinogenesis was first indicated by Ivankovic (1969) and Alexandrov (1973) They found that pregnant mice, when injected one or more doses of MNU, developed a high incidence of tumors of the uterus, vagina and breast cancer, however, when similar doses were administered in non-pregnant rats the results were different Schreiber et al (1972) found an increase in the number of extraneural tumors induced by MNU in rats to which previously had undergone ovariectomy However, neither the execution of ovariectomy, or the application of testosterone or other oral contraceptives, have altered the oncogenic results (Schreiber et al 1972; Thomas et al 1972)

Regarding the role of immunological factors in the development of nervous system tumors, there is very little data Delinger et al (1973) studied the effect of the suppression of cell-mediated immunity in carcinogenesis with MNU in Fischer rats They used a treatment with anti-lymphocyte serum and observed no change in the incidence of neurogenic tumors

3.3 Morphology and biology of nitrosourea-induced brain tumors

There are a number of compounds able to induce tumors in the nervous system However, all studies have been directed toward understanding the morphology of tumors induced by repeated doses of MNU in adult animals, or just for perinatal injection of ENU (Schiffer et al 1970; Koestner et al 1971; Lantos, 1972; Swenberg et al 1972, Jones et al 1973)

3.3.1 Tumor location

Regardless of the type of carcinogen used, preferably tumors develop in a number of specific regions of the nervous system For example, in the brain, they are located mainly in

the periventricular region, in the subcortical white matter of the cerebral hemispheres, and hippocampus The periventricular tumors usually develop around the lateral ventricles, including the caudate nucleus and corpus callosum Tumors rarely appear localized in the cerebellum In the spinal cord, are normally at cervical and lumbar segments These tumors are also developed on the cranial nerves, of which the trigeminal nerve is the most frequent location (Mennel and Zulch, 1971; Ivankovic, 1972, Schreiber et al 1972)

Gliomas of periventricular subependymal plate are the first tumors are the first tumors that develop, they are identical to the anaplastic glioependymomas, and almost equivalent to the periventricular pleomorphic gliomas originating from the undifferentiated cells of the subependymal plate described by Lantos (1972) The presence of true ependymomas between the ENU-induced tumors is controversial, and generally they have been considered

as such, either by their intraventricular location, or due to their histological features, reminiscent of ependymomatous tumors of humans Unequivocal ependymomas were not seen in series of mice exposed transplacentally to ENU, but according to accepted classifications, approximately 20% of the ENU-induced brain tumors could be diagnosed as ependymomas, anaplastic ependymomas, or mixed glial tumors with ependymoma areas

(Mandybur and Alvira, 1982) In many classifications, ependymomatous tumors were

termed as "anaplastic glioependymomas" due to the presence of ependymoma-like cells, but these tumor cells coexist with other glial-like cells, pleomorphic cells and generally with rounded cells being very similar to those of human oligodendrogliomas In any case, the histopathological diagnosis of the ENU-induced ependymomas is based on the existence of tumor cells arranged in rosettes around blood vessels Ultrastructurally, there are two cell types: a small undifferentiated cell, and a larger type, more differentiated Transitional forms between these two cell types can be seen Undifferentiated cells are small, with a relatively large nucleus and little cytoplasm The more differentiated tumor cells have a pleomorphic nucleus in an eccentric position, surrounded by abundant cytoplasm Overall neoplastic ependymal cells do not possess cilia or blepharoplasts, and are not equipped with junctional complexes Studies by Mandybur and Alvira (1982) supported that the named

“ENU-induced ependymomas” are not true ependymal tumors and that differ from the human ependymomas, because none of the ultrastructural features of normal or tumoral ependymal cells were present Therefore these authors suggested that these tumors may actually be regarded as undifferentiated tumors, with some features of ependymomas

On the other hand, the histology of tumors induced by ENU and MNU are similar There are however some differences that were highlighted by Swenberg et al (1972) These authors found that ENU-induced gliomas are better differentiated than the MNU-induced tumors, and that ENU produces a greater number of anaplastic schwannoma-like tumors In animals treated with MNU, gliosarcoma can be found in 10% of cases, however, this type of tumor is completely absent in the treatments with ENU, a carcinogen that produced almost exclusively oligodendroglioma-like tumors and malignant schwannoma-like tumors, as was pointed out by Schiffer et al (1970) This criterion has been confirmed in numerous studies later and most of the reviews about the morphology of the ENU-induced brain tumors reflected the observation that most tumors can be considered as malignant oligodendroglioma-like tumor or malignant schwannoma-like tumors (Vaquero et al, 1994) The oligodendroglioma-like tumors are characteristically located at the subcortical white matter of the cerebral hemispheres, showing macroscopic appearance of well-defined tumors, often with hemorrhagic characteristics and foci of necrosis Sometimes these tumors develop large cystic cavities

In light microscopy studies, the oligodendroglioma-like tumors show a fairly uniform cell population They are composed of small cells, which show a dark and small nucleus, and a clear cytoplasm Regressive changes are absent and there are small hemorrhagic foci Outlying areas of these tumors have a cellular isomorphism, which is not appreciated in the central areas, where the existing cell population shows more pleomorphism, containing giant cells, occasionally multinucleated Ultrastructural studies reveal the presence of neoplastic cells with an elongated or oval dark nucleus, and a small, clear cytoplasm, poor

in organelles However, some neoplastic cells show a dark nucleus and a dense cytoplasm These findings suggest that these tumors are primitive undifferentiasted tumors with some oligodendroglial features, and their undifferentiated character is supported by immunohistochemical studies On immunohistochemistry, there is a concordance between our results and those of other authors regarding the expression of the protein S-100, PGA and vimentin (Conley, 1979, Mauro et al 1983; Mennel and et al 1990; Raju, 1990; Reifenberg et al 1989) but importantly we have obtained strong synaptophysin positivity in most of these tumors Considering that in human pathology, this marker is useful for the recognition of primitive neuroectodermal tumors such as medulloblastoma (Molenaar et al, 1991) and also for the neuronal characterization of brain tumors, it is logical to suppose that the majority of ENU-induced brain tumors can be regarded as undifferentiated neuroectodermal tumors with possible neuronal differentiation, regardless of their morphological appearance Furthermore, in our studies, most of the ENU-induced oligodendroglioma-like tumors show immunopositivity to the neuroblastic marker NB-84 This finding agrees with some of the previous classifications of these neoplasms, such as that of Jones et al (1973), who first identified the ENU-induced tumors as neuroblastomas The schwannoma-like tumors generally developed at the skull base, on the zone of the Gasser ganglion They can be also located in the spinal root, with usually solid and sometimes cystic consistency In our studies, these malignancies began to show neurological symptoms after a latent period ranging between 3 and 7 months after carcinogen administration After 8 months of postnatal life, the development of these tumors is more

infrequent, and after this time, intracerebral neoplasms, mainly of oligodendroglioma-like type, started to become symptomatic

The microscopical study of these tumors with hematoxylin-eosin technique suggests that they can be classified "malignant schwannomas" They generally show a cell population highly isomorphous, consisting of small cells with dark and more or less rounded nucleus, usually in a central position, with the typical appearance of undifferentiated cells Furthermore, a great number of mitotic figures can be seen Moreover, in these tumors there

is a large blood supply, with hyperplasia of the vessels and the formation of large cystic spaces Sometimes is possible to find areas of necrosis Despite the large cellular isomorphism that characterizes these tumors, is possible see compact areas showing cells with fusiform aspect, arranged in palisade, or sometimes areas with looser reticular aspect When the tumors are located in the region of trigeminal ganglion, is frequent the presence of large neuron-like cells interspersed with the small undifferentiated cells, which supposedly correspond to trigeminal ganglion neurons that are trapped between tumor cells, but the possibility of actually correspond to a gangliocytic differentiation of the tumor can not be ruled out

In the ultrastructural study of these tumors, at least two cell types can be found On the one hand, there was a cell type with dark nucleus, whose chromatin is condensed to form a ring around the nuclear membrane and cytoplasmic features suggesting a neoplastic Schwann-cell The other cell type shows a small, dark and round nucleus usually with a central position and chromatin that was condensed at the nuclear periphery These cells showed a dense cytoplasm, with abundant rough endoplasmic reticulum, free ribosomes and polyribosomes, microtubules, primary lysosomes and a large quantity of mitochondria with dense matrix Some of these cells show cytoplasmic granular vesicles, suggesting neuronal differentiation

Fig 1 Macroscopic appearance of an ENU-induced intraparenchymatous brain tumor

Finally, interspersed with these two cell types, it is possible to observe the existence of small cells, with scant cytoplasm and much more irregular nuclear configuration, which were interpreted as undifferentiated tumor cells

The immunohistochemical study of these tumors shows a clear positivity for S-100 protein and synaptophysin Furthermore, neuroblastic specific markers, such as NB-84 are positive

in all cases Vimentin is strongly positive in only some cases, and finally, the detection of GFAP is negative in all cases

Fig 2 Microscopic aspects of ENU-induced brain tumors A: Tumor with oligodendroglial aspect showing abundant mitoses B: Expression of GFAP in astrocytes trapped in the tumor C: Expression of synaptophysin in an ENU-induced brain tumor with oligodendroglial appearance D: Tumor cells showing positivity to the neuroblastic marker NB-84

Fig 3 Macroscopic appearance of ENU-induced tumors at level of trigeminal ganglion (A) and lumbar roots (B)

Fig 4 A: Microscopic appearance of an ENU-induced tumor at level of trigeminal ganglion Mature neurons can be seen, generally interpreted as trapped neurons from trigeminal ganglion B: Ultrastructural aspect of tumor cells show undifferentiated aspect with dense granules (arrows), suggesting the neuroblastic nature of the tumor cells